The overall objectives of the project are to establish more clearly the role of acrosomal enzymes and microtubules during fertilization in the environment created by the oviduct. Our immediate goals are to establish the relationships between the proteolytic activity of the acrosome and the mechanisms by which these microtubule-like structures bind, transport, and/or release the proteolytic activity during penetration of the zona. We have now demonstrated that these structures exit at all stages of penetration of the zona pellucida during in vivo as well as in vitro fertilization, and that they have the same diameters as microtubules. The chemistry of these structures will be investigated to determine if they consist of alpha- and beta-tubulin subunits similar to other microtubules. Subcellular fractionation of rabbit and/or rhesus monkey sperm will be used to isolate the acrosomes, and analytical and preparatory electrofocusing will be used to isolate the alpha- and beta-tubulins. Amino acid analyses will be performed to establish their identity if sufficient quantities can be isolated. We will also attempt to obtain more detailed electron micrographs of these microtubule-like structures using in vivo fertilized rabbit ova. We have observed protofilaments in these tubules, and we will attempt to establish how many protofilaments are in each tubule, which will help to establish the identity of these structures. We also hope to establish if radial spokes, dynein arms, and central doublets are present during normal in vivo fertilization by electron microscopy. Another objective of this grant period will be to determine the effects of the secretory IgA inhibitor for acrosin on fertilization, and its hormonal control. At least one more major oviductal inhibitor for acrosin remains to be identified.